Name: a preclinical model of exertional heat stroke in mice
Uploaded: 2021-07-01T14:30:00
Description: Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

Este trabajo fue financiado por el Departamento de Defensa W81XWH-15-2-0038 (TLC) y BA180078 (TLC) y el BK and Betty Stevens Endowment (TLC). JMA fue apoyada por la ayuda financiera del Reino de Arabia Saudita. Michelle King estaba con la Universidad de Florida en el momento en que se realizó este estudio. Actualmente trabaja en el Gatorade Sports Science Institute, una división de PepsiCo R&D.

Todos los procedimientos han sido revisados y aprobados por la Universidad de Florida IACUC. Para el estudio se utilizan ratones machos o hembras C57BL / 6J, de ~ 4 meses de edad, con un peso dentro de un rango de 27-34 g y 20-25 g, respectivamente.
1. Implantación quirúrgica del sistema telemétrico de monitorización de temperatura
<ol><li>A su llegada del vendedor, permita que los animales descansen en el vivero durante al menos 1 semana antes de la cirugía para minimizar el estrés de...

<ol>
	<li>Leon, L. R., Bouchama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stroke.">Heat stroke.</a> Comprehensive Physiology. 5 (2), 611-647 (2015).</li><li>Laitano, O., Leon, L. R., Roberts, W. O., Sawka, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+exertional+heat+stroke+diagnosis,+prevention,+and+treatment.">Controversies in exertional heat stroke diagnosis, prevention, and treatment.</a> Journal of Applied Physiology. 127 (5), 1338-1348 (2019).</li><li>King, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+prior+illness+on+exertional+heat+stroke+presentation+and+outcome.">Influence of prior illness on exertional heat stroke presentation and outcome.</a> PLOS One. 14 (8), 0221329 (2019).</li><li>Carter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+hospitalizations+and+deaths+from+heat+illness+in+soldiers.">Epidemiology of hospitalizations and deaths from heat illness in soldiers.</a> Medicine and Science in Sports and Exercise. 37 (8), 1338-1344 (2005).</li><li>Howe, A. S., Boden, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-related+illness+in+athletes.">Heat-related illness in athletes.</a> The American Journal of Sports Medicine. 35 (8), 1384-1395 (2007).</li><li>Wallace, R. F., Kriebel, D., Punnett, L., Wegman, D. H., Amoroso, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+heat+illness+hospitalization+and+risk+of+early+death.">Prior heat illness hospitalization and risk of early death.</a> Environmental Research. 104 (2), 290-295 (2007).</li><li>Wang, J. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+heat+stroke+and+subsequent+cardiovascular+diseases.">The association between heat stroke and subsequent cardiovascular diseases.</a> PLOS One. 14 (2), 0211386 (2019).</li><li>Leon, L. R., Blaha, M. D., DuBose, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+cytokine,+corticosterone,+and+tissue+injury+responses+in+mice+during+heat+strain+recovery.">Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery.</a> Journal of Applied Physiology. 100 (4), 1400-1409 (2006).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 288 (1), 197-204 (2005).</li><li>Leon, L. R., Gordon, C. J., Helwig, B. G., Rufolo, D. M., Blaha, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory,+behavioral,+and+metabolic+responses+to+heatstroke+in+a+conscious+mouse+model.">Thermoregulatory, behavioral, and metabolic responses to heatstroke in a conscious mouse model.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 299 (1), 241-248 (2010).</li><li>King, M. A., Leon, L. R., Morse, D. A., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+cytokine+and+chemokine+responses+to+exertional+heat+stroke+in+mice.">Unique cytokine and chemokine responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 122 (2), 296-306 (2016).</li><li>Costa, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=l-Arginine+supplementation+prevents+increases+in+intestinal+permeability+and+bacterial+translocation+in+Male+Swiss+mice+subjected+to+physical+exercise+under+environmental+heat+stress.">l-Arginine supplementation prevents increases in intestinal permeability and bacterial translocation in Male Swiss mice subjected to physical exercise under environmental heat stress.</a> The Journal of Nutrition. 144 (2), 218-223 (2014).</li><li>Hubbard, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+in+the+heat+on+predisposition+to+heatstroke.">Effects of exercise in the heat on predisposition to heatstroke.</a> Medicine and Science in Sports. 11 (1), 66-71 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+acute+heatstroke+mortality.">Rat model of acute heatstroke mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 42 (6), 809-816 (1977).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+significance+of+selected+serum+enzymes+in+a+rat+heatstroke+model.">Diagnostic significance of selected serum enzymes in a rat heatstroke model.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 46 (2), 334-339 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+physical+effort+in+the+etiology+of+rat+heatstroke+injury+and+mortality.">Role of physical effort in the etiology of rat heatstroke injury and mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 45 (3), 463-468 (1978).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-dependent+responses+to+exertional+heat+stroke+in+mice.">Sex-dependent responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 125 (3), 841-849 (2018).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ibuprofen+during+Exertional+Heat+Stroke+in+Mice.">Effects of Ibuprofen during Exertional Heat Stroke in Mice.</a> Medicine and Science in Sports and Exercise. 52 (9), 1870-1878 (2020).</li><li>King, M. A., Leon, L. R., Mustico, D. L., Haines, J. M., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+multi-organ+injury+in+a+pre-clinical+model+of+exertional+heat+stroke.">Biomarkers of multi-organ injury in a pre-clinical model of exertional heat stroke.</a> Journal of Applied Physiology. 118 (10), (2015).</li><li>Murray, K. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+heat+stroke+leads+to+concurrent+long-term+epigenetic+memory,+immunosuppression+and+altered+heat+shock+response+in+female+mice.">Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.</a> The Journal of Physiology. 599 (1), 119-141 (2021).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 288, 197-204 (2005).</li><li>Laitano, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+metabolic+dysfunction+in+myocardium+following+exertional+heat+stroke+in+mice.">Delayed metabolic dysfunction in myocardium following exertional heat stroke in mice.</a> The Journal of Physiology. 598 (5), 967-985 (2020).</li><li>Iwaniec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+phase+response+to+exertional+heat+stroke+in+mice.">Acute phase response to exertional heat stroke in mice.</a> Experimental Physiology. 106 (1), 222-232 (2020).</li><li>He, S. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+rhabdomyolysis+model+in+mice+with+exertional+heat+stroke+mouse+model+of+EHS-rhabdomyolysis.">Optimization of a rhabdomyolysis model in mice with exertional heat stroke mouse model of EHS-rhabdomyolysis.</a> Frontiers in Physiology. 11, (2020).</li><li>Lopez, J. R., Kaura, V., Diggle, C. P., Hopkins, P. M., Allen, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+hyperthermia,+environmental+heat+stress,+and+intracellular+calcium+dysregulation+in+a+mouse+model+expressing+the+p.G2435R+variant+of+RYR1.">Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.</a> British Journal of Anaesthesia. 121 (4), 953-961 (2018).</li><li>Laitano, O., Murray, K. O., Leon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+mechanisms+of+exertional+heat+stroke+and+malignant+hyperthermia:+evidence+vs.+conjecture.">Overlapping mechanisms of exertional heat stroke and malignant hyperthermia: evidence vs. conjecture.</a> Sports Medicine. 50 (9), 115-123 (2020).</li><li>Casa, D. J., Armstrong, L. E., Kenny, G. P., O'Connor, F. G., Huggins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+heat+stroke:+new+concepts+regarding+cause+and+care.">Exertional heat stroke: new concepts regarding cause and care.</a> Current Sports Medicine Reports. 11 (3), 115-123 (2012).</li></ol>

Esta revisión técnica tiene como objetivo proporcionar pautas para el rendimiento de un modelo preclínico de EHS en ratones. Se proporcionan pasos detallados y materiales necesarios para la ejecución de un episodio de EHS reproducible de gravedad variable. Es importante destacar que el modelo imita en gran medida los signos, síntomas y disfunción multiorgánica observados en víctimas humanas de EHS11,19. Además, este modelo permite examinar el mecanismo s...

El golpe de calor se caracteriza por disfunción del sistema nervioso central y daño subsiguiente de los órganos en sujetos hipertérmicos1. Hay dos manifestaciones de golpe de calor. El golpe de calor clásico (CHS) afecta principalmente a las poblaciones de edad avanzada durante las olas de calor o a los niños abandonados en vehículos expuestos al sol durante los calurosos días de verano1. El golpe de calor por esfuerzo (EHS) ocurre cuando hay una incapacidad para termorregular adecuadamente durante el esfuerzo físico, típicamente, pero no siempre, bajo altas temperaturas ambientales que resultan en síntomas neurológicos, hipertermia y posterior disfunción y daño multiorgánico2. EhS ocurre en atletas recreativos y de élite, así como en personal militar y en trabajadores con y sin deshidratación concomitante3,4. De hecho, ehS es la tercera causa de mortalidad en atletas durante la actividad física5. Es extremadamente difícil estudiar ehS en humanos, ya que el episodio puede ser letal o conducir a resultados de salud negativos a largo plazo6,7. Por lo tanto, un modelo preclínico confiable de EHS podría servir como una herramienta valiosa para superar las limitaciones de las observaciones clínicas retrospectivas y asociativas en víctimas humanas de EHS. Los modelos preclínicos de CHS en roedores y cerdos han sido bien caracterizados8,9,10. Sin embargo, los modelos preclínicos de CHS no se traducen directamente en fisiopatología de EHS debido a los efectos únicos del ejercicio físico sobre el perfil termorregulador y la respuesta inmune innata11. Además, los intentos anteriores de desarrollar modelos preclínicos de EHS en roedores plantearon restricciones significativas, incluidos estímulos de estrés superpuestos inducidos por descarga eléctrica, inserción de una sonda rectal y temperaturas corporales centrales máximas predefinidas con altas tasas de mortalidad12,13,14,15,16 que no coinciden con los datos epidemiológicos actuales. Estos representan limitaciones significativas que pueden confundir la interpretación de los datos y proporcionar índices de biomarcadores poco confiables. Por lo tanto, el protocolo tiene como objetivo caracterizar y describir los pasos de un modelo preclínico estandarizado, altamente repetible y traducible de EHS en ratones que está en gran medida libre de las limitaciones mencionadas anteriormente. Se describen los ajustes al modelo que pueden resultar en resultados fisiológicos graduales de golpe de calor moderado a fatal. Según el conocimiento de los autores, este es el único modelo preclínico de EHS con tales características, lo que permite llevar a cabo investigaciones relevantes de EHS de una manera basada en hipótesis11,17,18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;1080P HD 4 Security Cameras 4CH Home Video Security Camera System w/ 1TB HDD 2MP Night View Cameras CCTV Surveillance Kit</td>
			<td>LaView</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Coated Vicryl Violet Braided</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Ethilon Nylon suture Black Monofilament</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Surgical Drape with Povidone 12x18</td>
			<td>Jorgensen Labset al.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BK Precision Multi-Range Programmable DC Power Supplies Model 9201</td>
			<td>BK Precision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DR Instruments&#160;Medical Student Comprehensive Anatomy Dissection Kit</td>
			<td>&#160;DR Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Energizer Power Supply</td>
			<td>Starr Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>G2 Emitteret al.</td>
			<td>Starr Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Layfayette Motorized Wheel Model #80840B</td>
			<td>Layfayette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Patterson Veterinary Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platform receiveret al.</td>
			<td>Starr Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Environmental Chamber Model 3911</td>
			<td>ThermoForma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Training Wheels</td>
			<td>&#160;Columbus Inst.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a preclinical model of exertional heat stroke in mice

El golpe de calor es la manifestación más grave de las enfermedades relacionadas con el calor. El golpe de calor clásico (CHS), también conocido como golpe de calor pasivo, ocurre en reposo, mientras que el golpe de calor por esfuerzo (EHS) ocurre durante la actividad física. EhS difiere de CHS en etiología, presentación clínica y secuelas de disfunción multiorgánica. Hasta hace poco, solo los modelos de CHS han sido bien establecidos. Este protocolo tiene como objetivo proporcionar pautas para un modelo de ratón preclínico refinado de EHS que esté libre de factores limitantes importantes como el uso de anestesia, restricción, sondas rectales o descargas eléctricas. En este modelo se utilizaron ratones machos y hembras C57Bl/6, instrumentados con sondas telemétricas de temperatura central (Tc). Para familiarizarse con el modo de carrera, los ratones se someten a 3 semanas de entrenamiento utilizando ruedas de carrera voluntarias y forzadas. A partir de entonces, los ratones corren en una rueda forzada dentro de una cámara climática establecida a 37.5 ° C y 40% -50% de humedad relativa (HR) hasta mostrar limitación de síntomas (por ejemplo, pérdida de conciencia) a Tc de 42.1-42.5 ° C, aunque se pueden obtener resultados adecuados a temperaturas de cámara entre 34.5-39.5 ° C y humedad entre 30% -90%. Dependiendo de la gravedad deseada, los ratones se retiran de la cámara inmediatamente para su recuperación a temperatura ambiente o permanecen en la cámara calentada durante más tiempo, lo que induce una exposición más grave y una mayor incidencia de mortalidad. Los resultados se comparan con los controles de ejercicio simulados (EXC) y/o los controles ingenuos (NC). El modelo refleja muchos de los resultados fisiopatológicos observados en el EHS humano, incluida la pérdida de conciencia, la hipertermia grave, el daño multiorgánico, así como la liberación de citoquinas inflamatorias y las respuestas de fase aguda del sistema inmunológico. Este modelo es ideal para la investigación basada en hipótesis para probar estrategias preventivas y terapéuticas que pueden retrasar la aparición de EHS o reducir el daño multiorgánico que caracteriza esta manifestación.

Los perfiles termorreguladores típicos durante la totalidad del protocolo EHS y la recuperación temprana de un ratón se ilustran en la Figura 1A. Este perfil comprende cuatro fases distintas que se pueden definir como la etapa de calentamiento de la cámara, la etapa de ejercicio incremental, la etapa de ejercicio en estado estacionario y una etapa de recuperación mediante un método de enfriamiento rápido (R) o severo (S)17. Los principales resultados termorregu...

Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

A Preclinical Model of Exertional Heat Stroke in Mice

El protocolo describe el desarrollo de un modelo preclínico estandarizado, repetible y de golpe de calor por esfuerzo (EHS) en ratones libres de estímulos externos adversos como la descarga eléctrica. El modelo proporciona una plataforma para estudios mecanicistas, preventivos y terapéuticos.

Un modelo preclínico de golpe de calor por esfuerzo en ratones

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Anteriormente, solo los modelos pasivos de golpe de calor han sido bien establecidos. Este modelo de golpe de calor por esfuerzo está libre de limitaciones importantes e imita más de cerca la fisiopatología humana. No es posible estudiar eficazmente el golpe de calor en humanos porque es una condición potencialmente mortal.Este modelo preclínico conserva las características clínicas del golpe de calor por esfuerzo humano. Esta técnica ayuda a probar los efectos de la velocidad de enfriamiento en la recuperación del golpe de calor por esfuerzo, las comorbilidades de la ingestión de drogas, así como las consecuencias a largo plazo de la exposición al golpe de calor. Demostrando el procedimiento estará Jamal Alzahrani, un estudiante de doctorado senior del laboratorio del Dr. Clanton.Después de anestesiar al ratón, use lubricante para los ojos como el ungüento veterinario para proteger los ojos del animal de daños o lesiones durante la cirugía. Para preparar el sitio quirúrgico, afeite la parte inferior del abdomen con cortapelos de animales pequeños o un removedor de pelo disponible comercialmente. Administrar la primera dosis de 0,1 miligramos subcutáneos por kilogramo de buprenorfina.Frote el área con tres lavados de povidona yodada o clorhexidina, seguido de un enjuague con alcohol isopropílico al 70%, luego transfiera el mouse al área quirúrgica, use una cortina adhesiva para aislar el sitio quirúrgico en el ratón. Usando instrumentos estériles en técnica aséptica, haga una incisión de aproximadamente un centímetro en la línea media a lo largo de la línea alba a unos 0,5 centímetros del margen costal, luego separe la piel de la capa muscular y haga una incisión ligeramente más pequeña en la línea alba teniendo cuidado de no dañar los intestinos u órganos internos. Una vez que la capa muscular esté abierta, coloque el telémetro estéril en la cavidad intraperitoneal frente a las arterias y venas caudales y dorsal a los órganos digestivos para permitir que flote libremente.Cierre la abertura abdominal con una sutura absorbible estéril de 5-O y cierre la piel con una simple sutura interrumpida con sutura de prolina 5-O. Coloque el ratón en su jaula limpia con una almohadilla térmica de microondas acurrucada debajo de la jaula. Monitoree al ratón cada 15 minutos durante la primera hora de recuperación de la anestesia y luego regrese a la instalación de alojamiento de animales.La noche antes del protocolo de golpe de calor por esfuerzo, coloque el ratón en la cámara ambiental a temperatura ambiente para aclimatarse a la cámara. Utilice el sistema de adquisición de datos para recopilar la temperatura central continua o TC promediada durante intervalos de 30 segundos durante la noche. En la mañana del protocolo de golpe de calor por esfuerzo, asegúrese de que el ratón esté en o por debajo del rango normal de temperatura diurna de 36 a 37.5 grados Celsius antes de aumentar la temperatura de la cámara para asegurarse de que el ratón no tenga fiebre o haya experimentado estrés indebido durante este período.Una vez que el ratón esté estable y dentro de un rango de temperatura central normal en reposo, retire la comida y el agua y pese al animal. Cierre la puerta de la cámara y aumente la temperatura de la cámara a un objetivo de 37,5 grados centígrados y de 40 a 50% de humedad relativa o la temperatura y humedad ambiental deseadas. Verifique la temperatura y la humedad de la cámara con un monitor de temperatura y humedad calibrado.Rodee la cámara con una cortina opaca para mantener la luz y las perturbaciones mínimas durante el protocolo. Supervise el ratón continuamente durante el protocolo a través de cámaras remotas iluminadas por infrarrojos. Enfoca la segunda cámara en el monitor de temperatura y humedad colocado cerca de la rueda de rodadura.Realice ajustes en el controlador para el punto de ajuste de la cámara ambiental para garantizar lecturas de temperatura precisas cerca del animal. Una vez que la cámara haya alcanzado su temperatura objetivo medida por la segunda cámara en el monitor de temperatura, abra rápidamente la puerta de la cámara y coloque el mouse en la rueda de carrera forzada. Inicie el protocolo de rueda de carrera forzada a una velocidad de 2,5 metros por minuto y aumente la velocidad en 0,3 metros por minuto cada 10 minutos hasta que el mouse alcance una temperatura central de 41 grados centígrados.Una vez que se haya alcanzado la temperatura central, permita que la velocidad permanezca constante hasta que se produzca la limitación de los síntomas, caracterizada por una aparente pérdida de conciencia, una caída hacia atrás o un desmayo, y la incapacidad de continuar corriendo o aferrándose a la rueda. Confirme el punto de tiempo en el que el ratón tiene tres rotaciones hacia atrás en la rueda sin signos de una respuesta física. Alternativamente, identifique el punto final humano siguiendo las reglas locales de IACUC para determinar cuándo detener el protocolo.Este criterio de valoración está ligeramente por encima de la limitación de los síntomas en prácticamente todos los ratones. Para realizar el protocolo de enfriamiento rápido, detenga la rueda una vez que el mouse alcance la limitación de síntomas y retire el mouse inmediatamente de la rueda de carrera forzada. Pesa el ratón y colócalo de nuevo en su jaula doméstica para recuperarlo a temperatura ambiente.Durante este tiempo, deje la puerta de la cámara abierta y devuelva el punto de ajuste de la incubadora a temperatura ambiente para permitir que la cámara se enfríe rápidamente, lo que resulta en una supervivencia a largo plazo superior al 99%. Para realizar una exposición más severa al golpe de calor por esfuerzo, mantenga la jaula del animal dentro de la cámara de 37.5 grados Celsius durante el protocolo de golpe de calor por esfuerzo. Cuando el animal alcance la limitación de los síntomas, permítale permanecer en la rueda de carrera hasta que vuelva a la conciencia según lo observado por la cámara remota, luego retire rápidamente el mouse de la rueda de carrera y devuélvalo directamente a su jaula precalentada para dar como resultado un perfil de enfriamiento mucho más lento, esencialmente eliminando la fase hipotérmica de golpe de calor por esfuerzo.Retire la parte superior del filtro de la jaula durante este tiempo para mejorar el equilibrio con la cámara. Se puede usar una jaula de recuperación preenfriada a temperatura ambiente para realizar un procedimiento alternativo menos severo para dar como resultado una fase hipotérmica suprimida con una tasa de supervivencia del 100% Para el protocolo de golpe de calor por esfuerzo severo, monitoree cuidadosamente a los ratones durante la recuperación y verifique continuamente los puntos finales humanos, incluidos los movimientos normales durante la recuperación, como el aseo, respiración normal y lamer. Controle la temperatura central durante este tiempo.Es poco probable que los ratones se recuperen si su temperatura central invierte la dirección durante la fase de recuperación, eventualmente superando los 40 grados centígrados. En tal caso, finalice el experimento y continúe evaluando el ratón para los puntos finales humanos estándar. Los perfiles termorreguladores típicos durante la totalidad del protocolo de golpe de calor por esfuerzo en la recuperación temprana de un ratón comprenden cuatro fases distintas que se pueden definir como la etapa de calentamiento de la cámara, la etapa de ejercicio incremental, la etapa de ejercicio en estado estacionario y una etapa de recuperación mediante un método de enfriamiento rápido o lento.Para el modelo severo, el perfil de temperatura de recuperación indicó que la temperatura central se mantuvo por encima de los 37 grados centígrados durante el período de recuperación de dos horas. Los ratones hembra eran más resistentes al golpe de calor en este modelo y corrían distancias casi dos veces más largas que los ratones machos. No se observaron diferencias en el tiempo requerido para recuperarse a 39,5 grados centígrados en los dos modelos.Sin embargo, el tiempo para enfriarse a la temperatura ambiental de 37,5 grados centígrados se prolongó considerablemente. Lo más importante es el seguimiento cuidadoso de los animales en todo momento post-cirugía y post-esfuerzo de golpe de calor. Este método ha ayudado a estudiar los efectos a largo plazo de la exposición al golpe de calor hasta varios meses, y también nos ha permitido medir los efectos de factores adicionales en la enfermedad por golpe de calor, como la ingestión de medicamentos antiinflamatorios no esteroideos y epigenética.

a-preclinical-model-of-exertional-heat-stroke-in-mice

Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferencia génica para la insuficiencia cardiaca isquémica en un modelo preclínico

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminal la arteria cerebral media de oclusión (MCAO) Modelo para el ictus isquémico con Orientación flujometría láser Doppler en ratones

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modelo murino de hemorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

A preclínicos modelo murino de metástasis hepática

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Carrera Modelado en ratones: Coagulación permanente de la arteria cerebral media Distal

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Un accidente cerebrovascular trombótico Basado En transitoria cerebral por hipoxia-isquemia Modelo

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Técnica quirúrgica para la oclusión de la arteria cerebral media y la reperfusión del movimiento inducidos en ratones

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Animal doméstico proyección de imagen de neuroinflamación usando [11C] DPA-713 en un modelo murino de ictus isquémico

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...